Exercise differentially affects metabolic functions and white adipose tissue in female letrozole- and dihydrotestosterone-induced mouse models of polycystic ovary syndrome.

Here we hypothesized that exercise in dihydrotestosterone (DHT) or letrozole (LET)-induced polycystic ovary syndrome mouse models improves impaired insulin and glucose metabolism, adipose tissue morphology, and expression of genes related to adipogenesis, lipid metabolism, Notch pathway and browning in inguinal and mesenteric fat. DHT-exposed mice had increased body weight, increased number of large mesenteric adipocytes. LET-exposed mice displayed increased body weight and fat mass, decreased insulin sensitivity, increased frequency of small adipocytes and increased expression of genes related to lipolysis in mesenteric fat. In both models, exercise decreased fat mass and inguinal and mesenteric adipose tissue expression of Notch pathway genes, and restored altered mesenteric adipocytes morphology. In conclusion, exercise restored mesenteric adipocytes morphology in DHT- and LET-exposed mice, and insulin sensitivity and mesenteric expression of lipolysis-related genes in LET-exposed mice. Benefits could be explained by downregulation of Notch, and modulation of browning and lipolysis pathways in the adipose tissue.

Adipose tissue macrophages impair preadipocyte differentiation in humans.

AIM: The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation. METHODS: Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified. RESULTS: Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001). With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance. CONCLUSIONS: The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

Adipose Cell Size and Regional Fat Deposition as Predictors of Metabolic Response to Overfeeding in Insulin-Resistant and Insulin-Sensitive Humans.

Obesity is associated with insulin resistance, but significant variability exists between similarly obese individuals, pointing to qualitative characteristics of body fat as potential mediators. To test the hypothesis that obese, insulin-sensitive (IS) individuals possess adaptive adipose cell/tissue responses, we measured subcutaneous adipose cell size, insulin suppression of lipolysis, and regional fat responses to short-term overfeeding in BMI-matched overweight/obese individuals classified as IS or insulin resistant (IR). At baseline, IR subjects exhibited significantly greater visceral adipose tissue (VAT), intrahepatic lipid (IHL), plasma free fatty acids, adipose cell diameter, and percentage of small adipose cells. With weight gain (3.1 ± 1.4 kg), IR subjects demonstrated no significant change in adipose cell size, VAT, or insulin suppression of lipolysis and only 8% worsening of insulin-mediated glucose uptake (IMGU). Alternatively, IS subjects demonstrated significant adipose cell enlargement; decrease in the percentage of small adipose cells; increase in VAT, IHL, and lipolysis; 45% worsening of IMGU; and decreased expression of lipid metabolism genes. Smaller baseline adipose cell size and greater enlargement with weight gain predicted decline in IMGU, as did increase in IHL and VAT and decrease in insulin suppression of lipolysis. Weight gain in IS humans causes maladaptive changes in adipose cells, regional fat distribution, and insulin resistance. The correlation between development of insulin resistance and changes in adipose cell size, VAT, IHL, and insulin suppression of lipolysis highlight these factors as potential mediators between obesity and insulin resistance.

Evidence for the regulatory role of lipocalin 2 in high-fat diet-induced adipose tissue remodeling in male mice.

Lipocalin 2 (Lcn2) has previously been characterized as an adipokine/cytokine playing a role in glucose and lipid homeostasis. In this study, we investigate the role of Lcn2 in adipose tissue remodeling during high-fat diet (HFD)-induced obesity. We find that Lcn2 protein is highly abundant selectively in inguinal adipose tissue. During 16 weeks of HFD feeding, the inguinal fat depot expanded continuously, whereas the expansion of the epididymal fat depot was reduced in both wild-type (WT) and Lcn2(-/-) mice. Interestingly, the depot-specific effect of HFD on fat mass was exacerbated and appeared more pronounced and faster in Lcn2(-/-) mice than in WT mice. In Lcn2(-/-) mice, adipocyte hypertrophy in both inguinal and epididymal adipose tissue was more profoundly induced by age and HFD when compared with WT mice. The expression of peroxisome proliferator-activated receptor-γ protein was significantly down-regulated, whereas the gene expression of extracellular matrix proteins was up-regulated selectively in epididymal adipocytes of Lcn2(-/-) mice. Consistent with these observations, collagen deposition was selectively higher in the epididymal, but not in the inguinal adipose depot of Lcn2(-/-) mice. Administration of the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (Rosi) restored adipogenic gene expression. However, Lcn2 deficiency did not alter the responsiveness of adipose tissue to Rosi effects on the extracellular matrix expression. Rosi treatment led to the further enlargement of adipocytes with improved metabolic activity in Lcn2(-/-) mice, which may be associated with a more pronounced effect of Rosi treatment in reducing TGF-ß in Lcn2(-/-) adipose tissue. Consistent with these in vivo observations, Lcn2 deficiency reduces the adipocyte differentiation capacity of stromal-vascular cells isolated from HFD-fed mice in these cells. Herein Rosi treatment was again able to stimulate adipocyte differentiation to a similar extent in WT and Lcn2(-/-) inguinal and epididymal stromal-vascular cells. Thus, combined, our data indicate that Lcn2 has a depot-specific role in HFD-induced adipose tissue remodeling.

Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.

Although erythropoietin (Epo) is the cytokine known to regulate erythropoiesis, erythropoietin receptor (EpoR) expression and associated activity beyond haematopoietic tissue remain uncertain. Here we show that mice with EpoR expression restricted to haematopoietic tissues (Tg) develop obesity and insulin resistance. Tg-mice exhibit a decrease in energy expenditure and an increase in white fat mass and adipocyte number. Conversely, Epo treatment of wild-type (WT)-mice increases energy expenditure and reduces food intake and fat mass accumulation but shows no effect in body weight of Tg-mice. EpoR is expressed at a high level in white adipose tissue and in the proopiomelanocortin (POMC) neurons of the hypothalamus. Although Epo treatment in WT-mice induces the expression of the polypeptide hormone precursor, POMC, mice lacking EpoR show reduced levels of POMC in the hypothalamus. This study provides the first evidence that mice lacking EpoR in non-haematopoietic tissue become obese and insulin resistant with loss of Epo regulation of energy homeostasis.

Cellularity and adipogenic profile of the abdominal subcutaneous adipose tissue from obese adolescents: association with insulin resistance and hepatic steatosis.

OBJECTIVE: We explored whether the distribution of adipose cell size, the estimated total number of adipose cells, and the expression of adipogenic genes in subcutaneous adipose tissue are linked to the phenotype of high visceral and low subcutaneous fat depots in obese adolescents. RESEARCH DESIGN AND METHODS: A total of 38 adolescents with similar degrees of obesity agreed to have a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test and hyperinsulinemic euglycemic clamp) and imaging studies (MRI, DEXA, (1)H-NMR). Subcutaneous periumbilical adipose cell-size distribution and the estimated total number of subcutaneous adipose cells were obtained from tissue biopsy samples fixed in osmium tetroxide and analyzed by Beckman Coulter Multisizer. The adipogenic capacity was measured by Affymetrix GeneChip and quantitative RT-PCR. RESULTS: Subjects were divided into two groups: high versus low ratio of visceral to visceral + subcutaneous fat (VAT/[VAT+SAT]). The cell-size distribution curves were significantly different between the high and low VAT/(VAT+SAT) groups, even after adjusting for age, sex, and ethnicity (MANOVA P = 0.035). Surprisingly, the fraction of large adipocytes was significantly lower (P < 0.01) in the group with high VAT/(VAT+SAT), along with the estimated total number of large adipose cells (P < 0.05), while the mean diameter was increased (P < 0.01). From the microarray analyses emerged a lower expression of lipogenesis/adipogenesis markers (sterol regulatory element binding protein-1, acetyl-CoA carboxylase, fatty acid synthase) in the group with high VAT/(VAT+SAT), which was confirmed by RT-PCR. CONCLUSIONS: A reduced lipo-/adipogenic capacity, fraction, and estimated number of large subcutaneous adipocytes may contribute to the abnormal distribution of abdominal fat and hepatic steatosis, as well as to insulin resistance in obese adolescents.

A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents.

UNLABELLED: The genetic factors associated with susceptibility to nonalcoholic fatty liver disease (NAFLD) in pediatric obesity remain largely unknown. Recently, a nonsynonymous single-nucleotide polymorphism (rs738409), in the patatin-like phospholipase 3 gene (PNPLA3) has been associated with hepatic steatosis in adults. In a multiethnic group of 85 obese youths, we genotyped the PNLPA3 single-nucleotide polymorphism, measured hepatic fat content by magnetic resonance imaging and insulin sensitivity by the insulin clamp. Because PNPLA3 might affect adipogenesis/lipogenesis, we explored the putative association with the distribution of adipose cell size and the expression of some adipogenic/lipogenic genes in a subset of subjects who underwent a subcutaneous fat biopsy. Steatosis was present in 41% of Caucasians, 23% of African Americans, and 66% of Hispanics. The frequency of PNPLA3(rs738409) G allele was 0.324 in Caucasians, 0.183 in African Americans, and 0.483 in Hispanics. The prevalence of the G allele was higher in subjects showing hepatic steatosis. Surprisingly, subjects carrying the G allele showed comparable hepatic glucose production rates, peripheral glucose disposal rate, and glycerol turnover as the CC homozygotes. Carriers of the G allele showed smaller adipocytes than those with CC genotype (P = 0.005). Although the expression of PNPLA3, PNPLA2, PPARγ2(peroxisome proliferator-activated receptor gamma 2), SREBP1c(sterol regulatory element binding protein 1c), and ACACA(acetyl coenzyme A carboxylase) was not different between genotypes, carriers of the G allele showed lower leptin (LEP)(P = 0.03) and sirtuin 1 (SIRT1) expression (P = 0.04). CONCLUSION: A common variant of the PNPLA3 gene confers susceptibility to hepatic steatosis in obese youths without increasing the level of hepatic and peripheral insulin resistance. The rs738409 PNPLA3 G allele is associated with morphological changes in adipocyte cell size.

Differential intra-abdominal adipose tissue profiling in obese, insulin-resistant women.

BACKGROUND: We recently identified differences in abdominal subcutaneous adipose tissue (SAT) from insulin-resistant (IR) as compared to obesity-matched insulin sensitive individuals, including accumulation of small adipose cells, decreased expression of cell differentiation markers, and increased inflammatory activity. This study was initiated to see if these changes in SAT of IR individuals were present in omental visceral adipose tissue (VAT); in this instance, individuals were chosen to be IR but varied in degree of adiposity. We compared cell size distribution and genetic markers in SAT and VAT of IR individuals undergoing bariatric surgery. METHODS: Eleven obese/morbidly obese women were IR by the insulin suppression test. Adipose tissue surgical samples were fixed in osmium tetroxide for cell size analysis via Beckman Coulter Multisizer. Quantitative real-time polymerase chain reaction for genes related to adipocyte differentiation and inflammation was performed. RESULTS: While proportion of small cells and expression of adipocyte differentiation genes did not differ between depots, inflammatory genes were upregulated in VAT. Diameter of SAT large cells correlated highly with increasing proportion of small cells in both SAT and VAT (r = 0.85, p = 0.001; r = 0.72, p = 0.01, respectively). No associations were observed between VAT large cells and cell size variables in either depot. The effect of body mass index (BMI) on any variables in both depots was negligible. CONCLUSIONS: The major differential property of VAT of IR women is increased inflammatory activity, independent of BMI. The association of SAT adipocyte hypertrophy with hyperplasia in both depots suggests a primary role SAT may have in regulating regional fat storage.

Guardian of corpulence: a hypothesis on p53 signaling in the fat cell.

Adipocytes provide an organism with fuel in times of caloric deficit, and are an important type of endocrine cell in the maintenance of metabolic homeostasis. In addition, as a lipid-sink, adipocytes serve an equally important role in the protection of organs from the damaging effects of ectopic lipid deposition. For the organism, it is of vital importance to maintain adipocyte viability, yet the fat depot is a demanding extracellular environment with high levels of interstitial free fatty acids and associated lipotoxic effects. These surroundings are less than beneficial for the overall health of any resident cell, adipocyte and preadipocyte alike. In this review, we discuss the process of adipogenesis and the potential involvement of the p53 tumor-suppressor protein in alleviating some of the cellular stress experienced by these cells. In particular, we discuss p53-mediated mechanisms that prevent damage caused by reactive oxygen species and the effects of lipotoxicity. We also suggest the potential for two p53 target genes, START domain-containing protein 4 (StARD4) and oxysterol-binding protein (OSBP), with the concomitant synthesis of the signaling molecule oxysterol, to participate in adipogenesis.

GLUT1 is not the primary binding receptor but is associated with cell-to-cell transmission of human T-cell leukemia virus type 1.

GLUT1 has recently been suggested to be a binding receptor for human T-cell leukemia virus type 1 (HTLV-1). We used a novel, short-term assay to define the role of GLUT1 in cell-to-cell transmission. Although increasing cell surface levels of GLUT1 enhanced HTLV-I transfer, efficient virus spread correlated largely with heparan sulfate proteoglycan (HSPG) expression on target cells. Moreover, since activated CD4+ T cells and cord blood lymphocytes that are susceptible to HTLV-1 infection expressed undetectable levels of surface GLUT1, these results indicate that GLUT1 and HSPGs are important for efficient cell-to-cell transmission of HTLV-1 but raise concerns on the role of GLUT1 as the HTLV-1 primary binding receptor.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 use different receptor complexes to enter T cells.

Studies using adherent cell lines have shown that glucose transporter-1 (GLUT-1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4(+) T cells, heparan sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT-1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4(+) T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8(+) T cells. Conversely, CD8(+) T cells, the primary target of HTLV-2, expressed GLUT-1 at dramatically higher levels than CD4(+) T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8(+) T cells while HTLV-1 SU binding and viral entry were higher on CD4(+) T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4(+) T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT-1 in CD4(+) T cells increased HTLV-2 entry, while expression of HSPGs on CD8(+) T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4(+) and CD8(+) T cells involved in entry.

Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF.

In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by approximately 50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.

7-hydroxystaurosporine (UCN-01) inhibition of Akt Thr308 but not Ser473 phosphorylation: a basis for decreased insulin-stimulated glucose transport.

7-hydroxystaurosporine (UCN-01) infused for 72 hours by continuous i.v. infusion induced insulin resistance during phase I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity with 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 mumol/L UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100,000 (100K) microunits/mL respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 mumol/L progressively shift the insulin dose-response curve even further. As Akt is known to mediate in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr308 phosphorylation in both an insulin and UCN-01 dose-dependent manner, whereas Akt Ser473 phosphorylation is inhibited only at the lowest insulin concentration, and then, only modestly. UCN-01 also inhibits insulin-induced Thr308 but not Ser473 phosphorylation of Akt associated with the plasma membranes and low-density microsomes and inhibits translocation of GLUT4 from low-density microsomes to plasma membranes as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr308 phosphorylation even in the face of almost completely unaffected Ser473 phosphorylation.

Evidence of impaired adipogenesis in insulin resistance.

To elucidate the roles of adipose tissue and skeletal muscle in the early development of insulin resistance, we characterized gene expression profiles of isolated adipose cells and skeletal muscle of non-diabetic insulin-resistant first-degree relatives of type 2 diabetic patients using oligonucleotide microarrays. About 600 genes and expressed sequence tags, which displayed a gene expression pattern of cell proliferation, were differentially expressed in the adipose cells. The differentially expressed genes in the skeletal muscle were mostly related to the cellular signal transduction and transcriptional regulation. To verify the microarray findings, we studied expression of genes participating in adipogenesis. The expression of Wnt signaling genes, WNT1, FZD1, DVL1, GSK3beta, beta-catenin, and TCF1, and adipogenic transcription factors, C/EBPalpha and beta and delta, PPARgamma, and SREBP-1, was reduced in the adipose tissue. The expression of adipose-specific proteins related to terminal differentiation, such as adiponectin and aP2, was reduced both in the adipose tissue and in the adipose cells isolated from portions of the biopsies. The adipose cells were enlarged in the insulin-resistant relatives and the cell size inversely correlated with the expression of the Wnt signaling genes, adiponectin, and aP2. Our findings suggest that insulin resistance is associated with an impaired adipogenesis.

Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.

Isolation and subsequent in vitro culture of primary adipose cells are associated with down-regulation of GLUT4 mRNA and simultaneous induction of GLUT1 gene expression. Progressive loss of insulin-responsive GLUT4 contributes to the decrease in insulin-mediated glucose uptake in these cells when cultured in vitro. The mechanisms underlying these alterations are unknown. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue triggers induction of many genes encoding inflammatory mediators including TNF-alpha, interleukin (IL)-1 alpha, IL-6, multiple chemokines, cell adhesion molecules, acute-phase proteins, type I IL-1 receptor, and multiple transcription factors implicated in the cellular inflammatory response. Secretion of TNF-alpha protein was also significantly induced during the 2-h collagenase digestion of adipose tissue. Isolated primary adipose cells exhibit dramatic changes in expression of multiple mRNAs that are characteristic of TNF-alpha-treated 3T3-L1 adipocytes including down-regulation of many genes important for insulin action and triglyceride synthesis. Addition of TNF-alpha to primary adipose cells in culture did not change the kinetics or the extent of the repression of adipose cell-abundant genes. Moreover, TNF-alpha-neutralizing antibody failed to block the changes in gene transcription in isolated primary adipose cells. Also, the standard isolation procedure induced the expression of NF-kappa B family members and their target genes in primary adipose cells prepared from TNF-alpha-/- mice to the same extent as in cells isolated from wild-type mice and resulted in almost identical changes in global gene expression when these cells were cultured in vitro. Thus, these data suggest that the standard isolation procedure-triggered reprogramming of gene expression in primary adipose cells that results in decreased insulin sensitivity does not require TNF-alpha, at least in this in vitro model system, but may be dependent on other inflammatory cytokines produced by these cells.

Adiponectin gene activation by thiazolidinediones requires PPAR gamma 2, but not C/EBP alpha-evidence for differential regulation of the aP2 and adiponectin genes.

We examined the role of PPAR gamma 2 and C/EBP alpha for adiponectin and aP2 gene activation in C/EBP alpha(-/-) fibroblasts by stably expressing PPAR gamma 2 or C/EBP alpha. PPAR gamma 2, but not PPAR gamma 1, mRNA markedly increased during the differentiation to adipocytes in cells expressing C/EBP alpha. Both infected cell lines differentiated to an adipocyte phenotype and the mRNA for both aP2 and adiponectin increased in parallel. However, adiponectin mRNA was considerably higher when C/EBP alpha was present, suggesting that this transcription factor is important for full gene activation. Thiazolidinediones markedly activated the gene in PPAR gamma 2-expressing cells in the absence of C/EBP alpha, suggesting that the adiponectin promoter may have functional PPAR gamma-response elements. Several observations showed that the adiponectin and aP2 genes can be differentially regulated in adipocytes: (1) Topiramate, an anti-epileptic agent with weight-reducing properties, increased adiponectin mRNA levels and secretion, but did not, like the thiazolidinediones, increase aP2 expression; (2) IL-6 reduced adiponectin, but significantly increased, aP2 expression; and (3) TNFalpha inhibited adiponectin, but paradoxically increased, aP2 expression in PPAR gamma 2-infected C/EBP alpha null cells. These data show that activation of the adiponectin gene can be separated from effects on adipogenic genes.

Activity, phosphorylation state and subcellular distribution of GLUT4-targeted Akt2 in rat adipose cells.

In this study, fusion of the kinase domain of Akt2 to the cytosolic C terminus of exofacially-HA-tagged GLUT4 is used to investigate the activity, phosphorylation state and subcellular localization of Akt2 specifically targeted to the GLUT4-trafficking pathway in rat adipose cells. Fusion of wild-type (wt) Akt2, but not a kinase-dead (KD) mutant results in constitutive targeting of the HA-GLUT4 fusion protein to the cell surface to a level similar to that of HA-GLUT4 itself in the insulin-stimulated state. Insulin does not further enhance the cell-surface level of HA-GLUT4-Akt2-wt, but does stimulate the translocation of HA-GLUT4-Akt2-KD. Cell-surface HA-GLUT4-Akt2-wt is found to be phosphorylated on Ser474 in both the absence and presence of insulin, and mutation of Ser474 to Ala reduces the increased basal cell-surface localization of the fusion protein. While Ser474 phosphorylation of HA-GLUT4-Akt2-KD is detected only in the insulin-stimulated state, trapping this fusion protein on the cell surface by coexpression of a dominant negative mutant dynamin does not induce Ser474 phosphorylation. Phosphorylation on Thr309 is not detectable in either HA-GLUT4-Akt2-wt or HA-GLUT4-Akt2-KD, in either the basal or insulin-stimulated state, and mutation of Thr309 to Ala does not influence the insulin-independent increases in cell-surface localization and Ser474 phosphorylation. Expression of HA-GLUT4-Akt2-wt stimulates the translocation of cotransfected myc-GLUT4 to a level similar to that in the insulin-stimulated state; this increase is moderately reduced by mutation of Ser474 to Ala and absent with the kinase-dead mutant. These results demonstrate that targeting Akt2 to the GLUT4-trafficking pathway induces Akt2 activation and GLUT4 translocation. Ser474 phosphorylation is an autocatalytic reaction requiring an active kinase, and kinase activity is associated with a plasma membrane localization. Fusion of Akt2 to the C terminus of GLUT4 appears to substitute for Thr309 phosphorylation in activating the autocatalytic process.